Cell Cycle and Cancer: The G1 Restriction Point and the G1 / S Transition

Expression of the small-subunit p49 mRNA of primase, the enzyme that synthesizes oligoribonucleotides for initiation of DNA replication, was examined in mouse cells stimulated to proliferate by serum and in growing cells. The level of p49 mRNA increased approximately 10-fold after serum stimulation and preceded synthesis of DNA and histone H3 mRNA by several hours. Expression of p49 mRNA was not sensitive to inhibition by low concentrations of cycloheximide, which suggested that the increase in mRNA occurred before the restriction point control for cell cycle progression described for mammalian cells and was not under its control. p49 mRNA levels were not coupled to DNA synthesis, as observed for the replication-dependent histone genes, since hydroxyurea or aphidicolin had no effect on p49 mRNA levels when added before or during S phase. These inhibitors did have an effect, however, on the stability of p49 mRNA and increased the half-life from 3.5 h to about 20 h, which suggested an interdependence of p49 mRNA degradation and DNA synthesis. When growing cells were examined after separation by centrifugal elutriation, little difference was detected for p49 mRNA levels in different phases of the cell cycle. This was also observed when elutriated G1 cells were allowed to continue growth and then were blocked in M phase with colcemid. Only a small decrease in p49 mRNA occurred, whereas H3 mRNA rapidly decreased, when cells entered G2/M. These results indicate that the level of primase p49 mRNA is not cell cycle regulated but is present constitutively in proliferating cells.

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E2F activity is regulated by cell cycle-dependent changes in subcellular localization.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.7268 ◽

1997 ◽

Vol 17 (12) ◽

pp. 7268-7282 ◽

Cited By ~ 142

Author(s):

R Verona ◽

K Moberg ◽

S Estes ◽

M Starz ◽

J P Vernon ◽

...

Keyword(s):

Cell Cycle ◽

Subcellular Localization ◽

Nuclear Localization ◽

Mammalian Cells ◽

Critical Role ◽

Biological Properties ◽

Dependent Manner ◽

Restriction Point ◽

Cycle Dependent ◽

Almost All

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. In this study, we show that the transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. We previously showed that E2F-4 accounts for the majority of endogenous E2F species. We now show that the subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed.

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A comprehensive model for the proliferation–quiescence decision in response to endogenous DNA damage in human cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715345115 ◽

2018 ◽

Vol 115 (10) ◽

pp. 2532-2537 ◽

Cited By ~ 29

Author(s):

Frank S. Heldt ◽

Alexis R. Barr ◽

Sam Cooper ◽

Chris Bakal ◽

Béla Novák

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Kinase Inhibitor ◽

Human Cells ◽

Cyclin Dependent Kinase ◽

Restriction Point ◽

Cyclin Dependent Kinase Inhibitor ◽

Serum Stimulation ◽

G1 Cells ◽

External Stimuli

Human cells that suffer mild DNA damage can enter a reversible state of growth arrest known as quiescence. This decision to temporarily exit the cell cycle is essential to prevent the propagation of mutations, and most cancer cells harbor defects in the underlying control system. Here we present a mechanistic mathematical model to study the proliferation–quiescence decision in nontransformed human cells. We show that two bistable switches, the restriction point (RP) and the G1/S transition, mediate this decision by integrating DNA damage and mitogen signals. In particular, our data suggest that the cyclin-dependent kinase inhibitor p21 (Cip1/Waf1), which is expressed in response to DNA damage, promotes quiescence by blocking positive feedback loops that facilitate G1 progression downstream of serum stimulation. Intriguingly, cells exploit bistability in the RP to convert graded p21 and mitogen signals into an all-or-nothing cell-cycle response. The same mechanism creates a window of opportunity where G1 cells that have passed the RP can revert to quiescence if exposed to DNA damage. We present experimental evidence that cells gradually lose this ability to revert to quiescence as they progress through G1 and that the onset of rapid p21 degradation at the G1/S transition prevents this response altogether, insulating S phase from mild, endogenous DNA damage. Thus, two bistable switches conspire in the early cell cycle to provide both sensitivity and robustness to external stimuli.

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Protein Phosphatases in G1 Regulation

International Journal of Molecular Sciences ◽

10.3390/ijms21020395 ◽

2020 ◽

Vol 21 (2) ◽

pp. 395 ◽

Cited By ~ 4

Author(s):

Ruth Martín ◽

Vilte Stonyte ◽

Sandra Lopez-Aviles

Keyword(s):

Cell Cycle ◽

Protein Phosphatases ◽

Eukaryotic Cells ◽

Signalling Pathways ◽

Cyclin Dependent Kinase ◽

Restriction Point ◽

Environmental Signals ◽

Substrate Phosphorylation ◽

Cell Cycle Machinery

Eukaryotic cells make the decision to proliferate, to differentiate or to cease dividing during G1, before passage through the restriction point or Start. Keeping cyclin-dependent kinase (CDK) activity low during this period restricts commitment to a new cell cycle and is essential to provide the adequate timeframe for the sensing of environmental signals. Here, we review the role of protein phosphatases in the modulation of CDK activity and as the counteracting force for CDK-dependent substrate phosphorylation, in budding and fission yeast. Moreover, we discuss recent findings that place protein phosphatases in the interface between nutritional signalling pathways and the cell cycle machinery.

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FAULTY CELL CYCLE RESTRICTION POINT G1/S IN HUMAN TESTICULAR TUMORS

The Journal of Urology ◽

10.1097/00005392-199904010-00638 ◽

1999 ◽

pp. 159

Author(s):

Bettina Schmidt ◽

Achim Rose ◽

Torsten Strohmeyer ◽

Rolf Ackermann

Keyword(s):

Cell Cycle ◽

Testicular Tumors ◽

Restriction Point ◽

Faulty Cell

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Identification of HiNF-P, a Key Activator of Cell Cycle-Controlled Histone H4 Genes at the Onset of S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8110-8123.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8110-8123 ◽

Cited By ~ 42

Author(s):

Partha Mitra ◽

Rong-Lin Xie ◽

Ricardo Medina ◽

Hayk Hovhannisyan ◽

S. Kaleem Zaidi ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Cyclin E ◽

S Phase ◽

Phase Cell ◽

Regulatory Sequences ◽

Histone H4 ◽

Restriction Point ◽

Histone H4 Gene

ABSTRACT At the G1/S phase cell cycle transition, multiple histone genes are expressed to ensure that newly synthesized DNA is immediately packaged as chromatin. Here we have purified and functionally characterized the critical transcription factor HiNF-P, which is required for E2F-independent activation of the histone H4 multigene family. Using chromatin immunoprecipitation analysis and ligation-mediated PCR-assisted genomic sequencing, we show that HiNF-P interacts with conserved H4 cell cycle regulatory sequences in vivo. Antisense inhibition of HiNF-P reduces endogenous histone H4 gene expression. Furthermore, we find that HiNF-P utilizes NPAT/p220, a substrate of the cyclin E/cyclin-dependent kinase 2 (CDK2) kinase complex, as a key coactivator to enhance histone H4 gene transcription. The biological role of HiNF-P is reflected by impeded cell cycle progression into S phase upon antisense-mediated reduction of HiNF-P levels. Our results establish that HiNF-P is the ultimate link in a linear signaling pathway that is initiated with the growth factor-dependent induction of cyclin E/CDK2 kinase activity at the restriction point and culminates in the activation of histone H4 genes through HiNF-P at the G1/S phase transition.

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Drug-Free Approach To Study the Unusual Cell Cycle of Giardia intestinalis

mSphere ◽

10.1128/msphere.00384-16 ◽

2017 ◽

Vol 2 (5) ◽

Cited By ~ 11

Author(s):

Kathleen Horlock-Roberts ◽

Chase Reaume ◽

Guillem Dayer ◽

Christine Ouellet ◽

Nicholas Cook ◽

...

Keyword(s):

Cell Cycle ◽

Drug Targets ◽

Giardia Intestinalis ◽

Centrifugal Elutriation ◽

Active Infection ◽

Intestinal Protozoa ◽

Restriction Point ◽

The World ◽

Drug Free ◽

Potential Drug Targets

ABSTRACT Giardias are among the most commonly reported intestinal protozoa in the world, with infections seen in humans and over 40 species of animals. The life cycle of giardia alternates between the motile trophozoite and the infectious cyst. The regulation of the cell cycle controls the proliferation of giardia trophozoites during an active infection and contains the restriction point for the differentiation of trophozoite to cyst. Here, we developed counterflow centrifugal elutriation as a drug-free method to obtain fractions of giardia cultures enriched in cells from the G1, S, and G2 stages of the cell cycle. Analysis of these fractions showed that the cells do not show side effects associated with the drugs used for synchronization of giardia cultures. Therefore, counterflow centrifugal elutriation would advance studies on key regulatory events during the giardia cell cycle and identify potential drug targets to block giardia proliferation and transmission. Giardia intestinalis is a protozoan parasite that causes giardiasis, a form of severe and infectious diarrhea. Despite the importance of the cell cycle in the control of proliferation and differentiation during a giardia infection, it has been difficult to study this process due to the absence of a synchronization procedure that would not induce cellular damage resulting in artifacts. We utilized counterflow centrifugal elutriation (CCE), a size-based separation technique, to successfully obtain fractions of giardia cultures enriched in G1, S, and G2. Unlike drug-induced synchronization of giardia cultures, CCE did not induce double-stranded DNA damage or endoreplication. We observed increases in the appearance and size of the median body in the cells from elutriation fractions corresponding to the progression of the cell cycle from early G1 to late G2. Consequently, CCE could be used to examine the dynamics of the median body and other structures and organelles in the giardia cell cycle. For the cell cycle gene expression studies, the actin-related gene was identified by the program geNorm as the most suitable normalizer for reverse transcription-quantitative PCR (RT-qPCR) analysis of the CCE samples. Ten of 11 suspected cell cycle-regulated genes in the CCE fractions have expression profiles in giardia that resemble those of higher eukaryotes. However, the RNA levels of these genes during the cell cycle differ less than 4-fold to 5-fold, which might indicate that large changes in gene expression are not required by giardia to regulate the cell cycle. IMPORTANCE Giardias are among the most commonly reported intestinal protozoa in the world, with infections seen in humans and over 40 species of animals. The life cycle of giardia alternates between the motile trophozoite and the infectious cyst. The regulation of the cell cycle controls the proliferation of giardia trophozoites during an active infection and contains the restriction point for the differentiation of trophozoite to cyst. Here, we developed counterflow centrifugal elutriation as a drug-free method to obtain fractions of giardia cultures enriched in cells from the G1, S, and G2 stages of the cell cycle. Analysis of these fractions showed that the cells do not show side effects associated with the drugs used for synchronization of giardia cultures. Therefore, counterflow centrifugal elutriation would advance studies on key regulatory events during the giardia cell cycle and identify potential drug targets to block giardia proliferation and transmission.

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